ࡱ> tvsa "jbjb11 $8[[```````,,,,@ih:,Rq`j``#jjj``jtD,````jj``\ |,H"90i6 j ,j`CHEM 8157 Bioanalytical Exercise 8 & 9 February 21, 2008 Craig, Courtney and Meng The Design of Genomic Tools for Gene GPX1 The gene chosen to work with in this experiment was GPX1. This gene encodes a member of the glutathione peroxidase family. Glutathione peroxidase functions in the detoxification of hydrogen peroxide, and is one of the most important antioxidant enzymes in humans. It has been reported that the protein encoded by this gene protects from CD95-induced apoptosis in cultured breast cancer cells and inhibits 5-lipoxygenase in blood cells, and its overexpression delays endothelial cell growth and increases resistance to toxic challenges. This protein is one of only a few proteins known in higher vertebrates to contain selenocysteine, which occurs at the active site of glutathione peroxidase and is coded by the nonsense (stop) codon TGA. FROM:  HYPERLINK "http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2876&ordinalpos=2&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum" http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2876&ordinalpos=2&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum PART I: Sequence of the gene From: http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=NT_022517.17&from=49333726&to =49336681&view=fasta): GCAGGGCATAGTCAGAAGGGCCAGTTAAATTATAGTTTTCTCACTCTTGAATATATGAGGCAGAAACTTGTAAGATCATCGTCACAGCAGATACAGGCACTTGGTAGCAACATGGAAAGATCTAGCCAGATAACAGAATTTATAACATTGCATGAGCTTCCGTGTAAAATGGGATTGTGTGGACCTAACAATTGGAGAGGCCATGACTCCAAATGTGAGTGGGGGCTCATGTTATTTTGTGACAGTCATTTCAGCGTGCAGCAAGTAGTCAGAAGTCTATCATTTGAGTTAGGCAGTCAACCATTGCCTTGTGTCTTTTTTGAAGTAAGGACTTTGTTGTAACTGGGTCTTGAGATGGCCCTTTGCATGTTCTGGTGAAGGCAGAAATGCAGGCCAGGAGCCAGACTTCCCACAAAGGAAAGAGTGGATCCTACACGCTGCAGACAAAAGGACAGAAAGTTCAGACTTGGCTAGGATAAGGGAAGGAGGATTAAGTTCCCACCACTATGACAATGAGGTTAGAAAAAGTATCGCCAGGCCGGGTGCGGTGGCTCATACCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGTGGATCACGAGGTCAGGAGATCGATACCATCCTGGCTAACACGGTGAAACCCTGTCTCTACTAAAAATACAAAAAATTAGCCGGGCGTGGTGGCAGGCACCTGTAGTCCCAGCTACTCAGGAGGTTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGTGAGCCTAGATTGCGCCACTGCACTCCAGCCTGGGCAATAGAGCAAAAAACTCCGTCTCAAAAAAAAAAAAAAAAAAAGAAAAGAAAAAGAATTCCCAAATGACAATGACACAGGACATACACACAGTTCTGCTGACACCCGGCACTTTATTAGTGGGGAAACTCGCCTTGGTCTGGCAGAGACTGGGATCAACAGGACCAGCACCCATCTCGAGGTGGTATTTTCTGTAAGATCAGGTGTTCCTCCCTCGTAGGTTTAGAGGAAACACCCTCATAGATGAAAACCCCCCCGAGACAGCAGCACTGCAACTGCCAAGCAGCCGGGGTAGGAGGGGCGCCCTAGGCACAGCTGGGCCCTTGAGACAGCAGGGCTTCGATGTCAGGCTCGATGTCAATGGTCTGGAAGCGGCGGCTGTACCTGCGTAGGGGCACACCGTCAGGGCCCACCAGGAACTTCTCAAAGTTCCAGGCAACATCGTTGCGACACACCGGAGACCAGGTGATGAGCTTGGGGTCGGTCATAAGCGCGGTGGCGTCGTCGCTGGGAGCTGGCAGGGCCTCCCGCAGGAAGGCGAAGAGAGGGTGCGCCCCCGCACCGTTCACCTCGCACTTCTCGAAGAGCATGAAGTTGGGCTCGAACCCACCACCAGGCCGGACGTACTTGAGGGAATTCAGAATCTCTTCGTTCTTGGCGTTCTCCTACAGGAGAGAAGGGCAGCTAGAACCCGGGGTCAAGAGGAGGAGAGAAACGTTCTAACCACAAACAAGGGAGATTTTCTATGAGTCACCGGGATTTTGCCCTCCATGCGCAATCCCAAGGGCGGAGAGGAATTTCAGCAGCTACGAGCAACAGAAAGGAAACGAGAGAGTAGCCAGACTCTCCGCGCATGGAGCCGACGGCACCCACCAGCACACCGCCGGCGCCCCCAGCCACTACTGCACGTCCGCCCCCGCCCCGCCCCGCTCCGCCCGGCGCACCTGATGCCCAAACTGGTTGCACGGGAAGCCGAGCACCACCAGGCCCCGGGGTCCGAGGCGCCGCTGCAGCTCGTTCATCTGGGTGTAGTCCCGGACCGTGGTGCCTCAGAGGGACGCCACATTCTCGATAAGTAGTACCTTGCCCCGCAGGGAGCCCAGGCTCACAGGCTCCCCGCCGGCCAGCGGGCGCGCCGAGAAGGCATACACCGACTGGGCCGCCGCCGCCGCCGCCGCTAGCCGAGCAGCACACATGGCGCAATTGTCCAAGAAGCCAGCGGAGCGCCCCGAACAAGCACTGTAAGGGGAGGCCAGCAGGCGCCTCCTTTTAACTGGCCGGCGGCGGGTCACGTGGCACAGGCAGTTTTCCGGGCCTGAGGGTCCCGCCTCATCCGGCCCCCGCCCTACCCGGAGGGCTCGGCCCCTCCTCAGCCAGGAAAAGGCTGGAGAGTGCGCCCGAGCGGGACGCGTGCTCCTCCTAAGCCGGAAGGAGCAGCAGGCATGTCTGGTCTGGCCCAGGCAACCAGGCGGCAGCGGTCCAGGTTACCCTTCCAAGGGCTGGGGCCCTCGAGGGCTCCGTTTTTTCTCAGAGGTTCCTGAGTTAGGATGTGGGGTTTGGCATAGGAGGCTGGTGGGCGACGGCAGCAGCTGCAGGAGCTGCCCGTGCACAAACAGCCCTCCTCTCCTGTGCCATGTTACCGGCAGTGCAGGGGACAGCCAGAGGGATCTAGGCTTCCGGTCTCCCTCCTCCCTGTCTCCAAAGCGGCACCGCCCTCGTGCGGGTTTTCTAACCGGGCAGAGTCCTGGAAGCTCTGCCCCAGCCTCCTTAAGTGGAGCCCAGTAGCTCGCCGAGGCCAAGGAAACGCTGCCGGAGTCCTCCCTCCCTGGCCTCCTCAGGCTGCATTCACCCTACCCCCAAATCTGAACCAGAATGCGGTACAGGGTAATCATTTCCATGTTGTTGGGAGGGACCAGATTAGCTGCTGGGTGGGTGGGAGGTGGGGCACAAGCTAGTCCTTTGCACTCAACAAACCTCTCCAGGG TGAGCTAGGCCAGGAAGTCCTTGCATCCTCCTACCCCCAGCCCTGGAGAGAGCAACACAAACAACAGGATACAGGAAGTTTAGAAAACTGCCTTTATTCTATTAGTAGTTGGAAAAATTAACTGGTACAGAAAAAAAGTTTAGTCAGCTGGAGAGAAGAGAGACTGAGTGCCACCCAT Restriction Enzyme: BstXI cuts the gene into three fragments as indicated above (black, red and black). The plasmid found with a restriction enzyme BstXI is pGEM-5Zf(+) (Cat.# P2241). If the gene, the plasmid and restriction enzyme are obtained and incorporated together, new plasmid containing the gene of interest can be cultivated. PART II: In order to monitor the expression of the glutathione peroxidase gene, real time PCR will be used to follow mRNA. A molecular beacon based RT-PCR will be used. The greater the fluorescence detection, the higher the mRNA concentration and thus a greater amount of gene expressed.  This 25 base molecular beacon binds to the gene starting at base 1568. Once emitter and quencher are separated, fluorescence is no longer quenched and can be detected. PART III: Scenario 1: Nick Translation Using the sequence of the gene of interest, a complementary DNA strand is used to conduct a FISH (fluorescence in situ hybridization) experiment. The first step in a FISH experiment is the random nicking of the bases on the gene using DNase. The addition of biotin-labeled nucleotide triphosphate bases, in excess, will promote repair with the fluorescent bases. The strand is then denatured with heat to separate the strands. The strand of interest is then hybridized to a glass microscope slide. An avidin antibody (which binds tightly to biotin) that is linked with a fluorophor will cause fluorescence that is seen under epifluorescent microscopy. The gene will then be located. Biotin kits are available from Invitrogen, and should be chosen based on the length of the sample strand. Scenario 2: Using PCR The making of the probe can also be done using the mRNA from the RT-PCR experiment above. RT-PCR will continually split and amplify, until an appropriate size is reached. The strands then need to be separated, and only one will be in used as a template to make the probe. Instead of adding excess phosphorylated nucleotides, amine-modified nucleotides are incorporated into the sequence. This sequence is then labeled, using AlexaFluor dyes or others on the market. Again, fluorescence microscopy can be employed to detect the position of the gene. AlexaFluor kits are available from Invitrogen. Some things that should be noted: These two examples would likely be interphase preparations, as a whole chromosome is not to be used. Additionally, choosing an appropriate method from the above two depends on sequence size PCR should be used in preparing smaller strand fragments for FISH. Nick translation would work better for larger fragments. 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